Purification of luo han guo extract

ABSTRACT

A method of purifying a Luo Han Guo extract includes contacting the Luo Han Guo extract with activated carbon and a macroporous polymeric adsorbent resin, an ion exchange resin, or both.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/215,647, filed Mar. 17, 2014, which claims priority from U.S.application Ser. No. 13/355,852, filed Jan. 23, 2012, now U.S. Pat. No.8,962,698, issued Feb. 24, 2015, which claims priority from U.S.provisional application 61/437,399, filed Jan. 28, 2011, the entirecontents of which are hereby incorporated herein by reference for allpurposes.

BACKGROUND

Natural caloric sweeteners, such as sucrose, glucose, and fructose,possess desirable taste characteristics, but they add to the caloriccontent of products. Therefore, there is great consumer interest in lowor non-caloric sweeteners that are considered as healthier alternatives.Non-caloric natural and synthetic high-potency sweeteners are known, butthey most often possess flavor profiles that are not as desirable toconsumers as sugars. Thus, it is desirable to develop non-caloricsweeteners that can be substituted for sugar and that have a moredesirable taste profile.

The species Stevia rebaudiana (“Stevia”) is the source of certainnaturally occurring sweet steviol glycosides. Considerable research anddevelopment has been done to evaluate the use of sweet steviolglycosides of Stevia as non-caloric sweeteners. Sweet steviol glycosidesthat may be extracted from Stevia include the six Rebaudiosides (i.e.,Rebaudiosides A to F), stevioside (the predominant glycoside in extractsfrom wild type Stevia), steviolbioside, rubusoside, and dulcosides.

Commercial low or non-caloric sweeteners based on Rebaudioside A andother sweet steviol glycosides tend to have bitter and liquoriceaftertastes. These characteristics are especially notable atconcentrations above about 300 ppm. In food applications, preferred uselevels (8-10% sugar equivalence values) are typically about 500 ppm toabout 1000 ppm, above the range at which off tastes are first noticed.Thus a need continues to exist for reduced-, low-, and/or non-caloricsweeteners including sweet steviol glycosides that have taste profileswith reduced or no bitterness, undesirable flavors (e.g., licorice), orsweetness profiles more like natural caloric sweeteners, or combinationsof such properties.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition including MogrosideV and a Rebaudioside component in a weight ratio ≧1:1 and ≦6:1, whereinthe Rebaudioside component consists of one or more compounds selectedfrom the group consisting of Rebaudioside A, Rebaudioside B andRebaudioside D.

In another aspect, the invention provides a method of purifying a LuoHan Guo extract that includes contacting the Luo Han Guo extract withactivated carbon and a macroporous polymeric adsorbent resin, an ionexchange resin, or both.

In yet another aspect, the invention provides a composition including aLuo Han Guo extract, wherein Mogroside V constitutes from 50 wt % to 75wt % of the Luo Han Guo extract and the composition includes from 0 to13 wt % in total relative to the Mogroside V of aromatic glycosides, andfrom 0 to 15 ppm of semi-volatile organic compounds relative to theMogroside V.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HPLC analysis of an exemplary dry Luo Han Guo extract, andanalysis of the same material that had been carbon treated according tothe invention, in the upper and lower chromatograms respectively.

FIG. 2 shows enlarged views of the chromatograms shown in FIG. 1.

FIG. 3 shows gas chromatograms of semi-volatile organic compoundspresent in a sample of Luo Han Guo, one taken before treatment withactivated carbon and one after treatment with activated carbon accordingto the invention.

FIG. 4 shows an HPLC chromatogram of a fraction of Luo Han Guocontaining components producing a musty flavor.

FIG. 5 shows an ATR-FTIR spectrum of a Luo Han Guo fraction showingcharacteristic bands consistent with the presence of an aromaticglycoside.

FIG. 6 shows a Time-of-Flight (ToF) accurate Mass Spectrum for the majorcomponent in FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the phrase “sweet steviol glycoside” means any naturallyoccurring compound having the general structure of a steviol diterpenering system with one or more saccharide residues chemically attached tothe ring.

As used herein, the phrase “Rebaudioside component” means the total ofRebaudioside A, B, and D present, with the understanding that only oneor two of these may in fact be present.

Sweetening Compositions Including Rebaudioside-Mogroside V Blends

It is now disclosed that blends of Mogroside V with a Rebaudiosidecomponent consisting of one or more of Rebaudiosides A, B and D providesuperior flavor characteristics, in many cases superior to either theRebaudioside component or the Mogroside V alone, when compared at anequal level of sweetness. In some systems, the improved taste is mostevident at pH values from about pH 2 to about pH 8.

Mogroside V may be obtained from extracts of Luo Han Guo, availablecommercially from a number of sources. Exemplary methods of producingsuch extracts are described in U.S. Pat. No. 5,411,755 and U.S. Publn.No. 2006/0003053, both incorporated herein by reference for all usefulpurposes. Luo Han Guo is extracted from the fruit of Siraitiagrosvenorii, an herbaceous perennial vine native to southern China andNorthern Thailand. It is one of four species in the genus Siraitia.Botanical synonyms include Momordica grosvenorii and Thladianthagrosvenorii. The extract is approximately 200-300 times as sweet assucrose.

Typically, Mogroside V is the most abundant single Mogroside componentof Luo Han Guo extracts, accompanied by other Mogrosides such asMogrosides I, II, III, IV and VI as well as other extracted materials,such as polyphenols, flavonoids, melanoidins, terpenes, proteins,sugars, aromatic glycosides, and semi-volatile organic compounds. Insome embodiments of the invention, the Mogroside V is provided in theform of a Luo Han Guo extract (either raw or purified and/orconcentrated to increase Mogroside V content). In some embodiments,Mogroside V constitutes at least 40 wt % of the extract, or at least 45wt %, or at least 50 wt %. Typically, it will constitute at most 95 wt %of the extract, at most 85 wt % of the extract, at most 75 wt % of theextract, at most 70 wt % of the extract, or at most 65 wt %, or at most60 wt %.

In some sweetening compositions according to the invention, the weightratio of Mogroside V to the Rebaudioside component is at least 1:1, orat least 1.3:1, or at least 1.5:1. The weight ratio is typically at most5:1, or at most 4:1, or at most 3.5:1, or at most 3:1, or at most 2.5:1,or at most 2:1, or at most 1.9:1, or at most 18:1, or at most 1.7:1.

The Rebaudioside component consists of one or more of Rebaudioside A, Band/or D. The Rebaudioside component typically constitutes at least 65wt % of the total sweet steviol glycosides present, or at least 70 wt %,or at least 75 wt %, or at least 80 wt %, or at least 90 wt %, or atleast 97 wt %. The balance of sweet steviol glycosides, if any, mayinclude one or more of Rebaudiosides C, E and/or F, stevioside, and anyother sweet steviol glycoside not part of the Rebaudioside component.Typically, Rebaudioside A will constitute at least 50 wt % of the sweetsteviol glycosides present, or at least 60 wt %, or at least 70 wt %, orat least 80 wt %, or at least 90 wt %, or at least 95 wt %. RebaudiosideA, B and D may be obtained from extracts of Stevia rebaudiana, availablecommercially from a number of sources. Many different methods ofproducing such extracts and obtaining relatively pure Rebaudioside A, Bor D from the extracts are known and have been described in theliterature, in one typical process, stevia plants are dried andsubjected to a water extraction process. This crude extract containsabout 50% Rebaudioside A. The various glycoside molecules in the extractare separated via crystallization techniques, typically using ethanol ormethanol as solvent, permitting the isolation of pure Rebaudioside A, Band D. The individual purified glycosides may then be used incombination to provide Rebaudioside components useful in the presentinvention.

Although sweetening compositions of the invention may include mixturesof various types of sweeteners in various quantities, in someembodiments the composition consists essentially of an optionallypurified and/or concentrated Luo Han Guo extract and an optionallypurified and/or concentrated Stevia extract.

Removal of Off-Flavor Components of Luo Han Guo

It has now also been found, after extensive studies, that the presenceof certain impurities in Luo Han Guo extracts results in an off-flavordescribed by some taste testers as “musty”. In particular, aromaticglycosides and semi-volatile organic compounds have been identified asproducing this undesirable flavor, although additional musty or otheroff-flavor components may also be present. One particular aromaticglycoside has a molecular mass of 502 Daltons, and appears to beparticularly productive of the musty flavor. This compound is accordingto the formula C₂₆H₃₀O₁₀, and all compounds in total having thismolecular formula are in some embodiments limited according to theinvention. Any means of achieving a sufficiently low level of thiscompound is suitable for purposes of the invention. One suitable way isto pass an aqueous solution of the Mogroside V, for example in the formof an optionally purified and/or concentrated Luo Han Guo extract,through a column of granular activated carbon. Other forms of activatedcarbon, for example powders, may also be used. The carbon treatment alsotypically removes additional musty or other off-flavor components aswell as pesticide residues and other such substances which are generallyundesirable in ingredients intended for human consumption. Typically,water is the only carrier present during the carbon treatment, and noorganic solvents are added. In some embodiments, the Luo Han Guo extractis treated with a macroporous polymeric adsorbent resin, an ion exchangeresin, and the activated carbon. Typically the treatments will be inthat order, but they need not be. One exemplary macroporous polymericresin is available commercially from Rohm and Haas, Philadelphia, Pa.under the trade name AMBERLITE® XAD1180N. An exemplary suitable ionexchange resin is an anionic resin is available under the trade nameAMBERLITE® FPA90 CL, also from Rohm and Haas.

The treatment must employ a sufficient amount of activated carbon, andmust occur with a sufficiently long contact time, to reduce the level ofthe one or more aromatic glycoside and semi-volatile organic compoundimpurities to an acceptable level. In some embodiments of the inventionthe sweetening composition comprises from 0 to 13 wt % in total ofaromatic glycosides, or from 0 to 11 wt %, or from 0 to 10 wt %, or from0 to 9 wt %, all relative to Mogroside V. The aromatic glycosides may bephenyl glycosides or more specifically phenolic glycosides, or they maybe coumarin glycosides or more specifically furanocoumarin glycosides.These same limits may also be appropriate in some embodiments forcompounds of molecular mass 502, and more specifically for compoundsaccording to the formula C₂₆H₃₀O₁₀, in each case referring to the totalamount of all compounds of mass 502 or of formula C₂₆H₃₀O₁₀.

In some embodiments of the invention the sweetening compositioncomprises from 0 to 15 ppm wt in total of semi-volatile organiccompounds, or from 0 to 11 ppm wt, or from 0 to 7 ppm wt, or from 0 to 3ppm wt, all relative to Mogroside V. The term “semi-volatile” as usedherein means compounds having a molecular weight in excess of 120Daltons and a boiling point at 1 atm pressure greater than 150° C. andup to 350° C. Such semi-volatile organic compounds may comprise, but arenot limited to, the compounds listed in Table 7. The semi-volatileorganic compounds may for example include aliphatic furans, unsaturatedaliphatics, esters, polycyclic hydrocarbons and/or terpenoids.

Commercially available Luo Han Guo powdered fruit extract, typicallycontaining at least 40% of Mogroside V (d.s.b), may be treated withactivated carbon as follows. Dry extract is dissolved in deionized waterat a concentration of at least about 1 wt %, and typically at most about70 wt %. The water is heated to a temperature sufficient to favor thedissolution of the powdered material, typically in a range betweenambient temperature and 160° F. (71.1° C.), and optionally filteredusing a microfiltration membrane or using filtration paper with a nonreactive filtration aid. The purpose of the microfiltration is to removeinsoluble proteins and/or microorganisms that could deteriorate theproduct. The resulting filtrate is subjected to adsorption with activecarbon (also known as activated carbon). The carbon may be any form ofactive carbon available, and may for example be derived from wood,bituminous coal, lignite coal, coconut, bone char, or any other source.In one embodiment, the active carbon is obtained by steam activation ofcarbon from lignite coal. Typically, the carbon is in the form ofgranules, but other physical forms such as powders or bead activatedcarbon may also be employed. It will generally be advantageous toutilize an active carbon which is highly porous and which has a highsurface area (e.g., over 100 m²/g, over 200 m²/g, or over 300 m²/g). Thenon-desirable components causing the off-taste (as well as otherundesirable substances such as pesticides) are adsorbed to the carbon,but the improved taste material is not adsorbed and is continuouslyeluted. The method allows for recovery yields (dry substance basis)between 50% and 99.9%. The amount of active carbon used may vary from0.05% to 150% (as a percentage of the dry substance present in theaqueous solution of Luo Han Guo fruit extract). More typically, toachieve sufficiently low levels of off-taste components, at least 2 wt %or at least 5 wt % of activated carbon relative to Luo Han Guo fruitextract is used on a solids basis. In certain embodiments, at least 6 wt% or at least 10 wt % gives the best results. Typically, at most 15 wt %will be used.

In a typical process, a column is packed with the desired amount ofactive carbon (typically in granulated form), and deionized water is runthrough the column from top to bottom or bottom to top (downflow orupflow direction) at a flow rate that ranges from 1 to 10 bed volumesper hour. The amount of water to pass could vary from 2 to 5 bedvolumes. Once the water has displaced the remaining air and some fineparticulates from the carbon, the aqueous solution of the Luo Han Guofruit extract is fed to the column at a flow rate that could range from1 to 10 bed volumes per hour. The column should be jacketed and thejacket temperature should be maintained at the same temperature as thefeed solution, which will typically be in a range from room temperatureto 71° C. Initially, the feed displaces the water in the column. Oncethe column effluent shows signs of material present, the effluent iscollected as improved taste material. The presence of solids in theeffluent can be assessed by measurement of the refractive index (RI). Acorrelation between RI and dry substance is typically built for thispurpose.

The Luo Han Guo fruit extract is fed to the column until the targetedtreatment level has been reached. Once the feed ends, the remaining LuoHan Guo fruit extract still present in the column is chased withdeionized or reverse osmosis water, displacing the Luo Han Guo material.The effluent collection is continued until the refractive index of theeffluent is close to that of water alone.

Optionally, the recovered improved taste material can be concentrated inorder to increase the DS % (dry substance) to any suitable level forsubsequent drying, if desired. The concentration can be completed byevaporation or membranes, or by any other suitable method. Membraneconcentration is possible with utilization of a nanofiltration membrane(200 Da. M.W.C.O.) or with a reverse osmosis membrane (with a saltrejection assay >98%). Both membranes can be used separately withoutlosing a significant amount of mogrosides to the permeate. The materialis then dried by using a conventional spray drying unit or by using aconventional spray agglomeration unit, or other means. Or the materialmay be used as-is. In one embodiment, the recovered improved tastematerial is combined with one or more other components, such asRebaudioside A, B and/or D or a purified Stevia extract containingcomprising sweet steviol glycosides, prior to drying.

Use of Sweetening Compositions Including Rebaudioside-Mogroside V Blends

Compositions containing Rebaudioside-Mogroside V blends may be processedusing known methods to modify particle size and physical form. Methodssuch as agglomeration, spray-drying, drum drying and other forms ofphysical processing may be applied to adjust particle size in order todeliver better flow, hydration, or dissolution properties. Thecompositions may be provided in liquid forms, optionally containing oneor more preservatives and/or processing aids, for ease-of-use inspecific applications. Compositions containing Rebaudioside-Mogroside Vblends may be co-processed with bulking agents such as maltodextrins andsimilar compounds to deliver products with controlled sweetness, dosing,potency, and handling properties.

Sweetening compositions of the present invention are useful asreduced-caloric, low-caloric, or non-caloric sweeteners in foodstuffs,i.e., edible or chewable compositions such as food, beverages, medicine,candy, chewing gum, and the like. It has been discovered that thesweetening compositions of the present invention can possess a sweetnessprofile that is more sugar-like and has reduced bitter aftertaste andreduced off-flavors (e.g., licorice) than sweeteners including onlysweet steviol glycosides. Testing has shown that, in most cases,sweetening compositions of the present invention are preferred by testsubjects over compositions that include 97% Rebaudioside A, when testedat a concentration providing equal sweetness. In particular, thesweetening compositions provide both immediate sweetness and delayedsweetness, resulting in a more satisfying flavor. Adding sweeteningcompositions of the present invention to foods and beverages is expectedto result in better tasting foods and beverages compared to thoseprepared with known sweetening composition containing sweet steviolglycosides, such as compositions having 97% Rebaudioside A as thesweetener.

Sweetener compositions according to the invention may include, inaddition to the Rebaudioside-Mogroside V blend, other high potencysweeteners. For example, sweet steviol glycosides may be included.Specific examples of suitable high potency sweeteners include naturalhigh potency sweeteners such as:

-   -   dulcoside A, dulcoside B (also known as Rebaudioside C),        rubusoside, mogroside III, mogroside IV, mogroside VI,        siamenoside, monatin and its salts (monatin SS, RR, RS, SR),        curculin, glycyrrhizic acid and its salts, thaumatin, mabinlin,        brazzein, hernandulcin, phyllodulcin, glycyphyllin, and        phloridzin;        and artificial high potency sweeteners such as:    -   saccharin, aspartame, sucralose, neotame, cyclamate and        acesulfame potassium.

According to the invention, Rebaudioside-Mogroside V blends may also becombined with caloric sweeteners such as sugars (e.g., high fructosecorn syrup, sucrose, fructose, etc.) and polyols (e.g., sorbitol,xylitol, lactitol, etc.) and/or other low-calorie sweeteners to producesweetening compositions of reduced caloric value.

In some embodiments, the invention provides foodstuffs includingsweetening compositions with high concentrations ofRebaudioside-Mogroside V blends. Essentially any edible or chewablecomposition may be sweetened in accordance with the invention.Nonlimiting examples include foodstuffs, (e.g., baked goods, soups,sauces, processed meats canned fruits, canned vegetables, dairyproducts, frozen confections); beverages (e.g., carbonated soft drinks,ready to drink teas, sports drinks, dairy drinks, alcoholic beverages,energy drinks, flavored waters, vitamin drinks, fruit drinks, and fruitjuices, powdered soft drinks), medicines or pharmaceutical products(e.g., tablets, lozenges, suspensions, etc.), nutraceutical products(e.g., supplements, vitamins, etc.), candy or confections; chewing gum;tobacco products (e.g., chewing tobacco); and the like. The sweeteningcomposition is included in an amount effective to impart the desiredamount of sweetness to the sweetened product. In some embodiments, thepH of the sweetened product is at least about 2 and not greater thanabout 8.

In some embodiments, the foodstuff contains a sweetening compositionincluding Rebaudioside-Mogroside V blends and one or more additionalsweet steviol glycosides as described herein. In some embodiments, thesweetening composition inclusive of the Rebaudioside component,additional steviol glycosides components, and the Mogroside V componentis present in the foodstuff at a total concentration of at least about50 ppm, or at least about 200 ppm, or at least about 500 ppm, or atleast about 1000 ppm, or at least about 1500 ppm, or at least about 3500ppm, or at least about 5000 ppm.

EXAMPLES Example 1 Stevia-Mogroside V Blends Vs. Rebaudioside APreference Testing

Blends of a solid Luo Han Guo extract containing 50 wt % Mogroside Vwith a Stevia product containing mostly Rebaudioside A were compared insweetness and preference panel testing against 97 wt % Rebaudioside. TheLuo Han Guo extract was a purified version of a commercial productavailable from Biovittoria (Guilin, People's Republic of China) underthe trade name Fruit Sweetness™, where the purification had been bytreatment with activated carbon as described elsewhere herein to removearomatic glycosides and semi-volatile organic compounds, which produceoff-flavors. This product is identified below as Sample A. The Steviaproduct was a commercial product available from GLG Life TechCorporation of Vancouver, B.C., Canada under the trade name BlendSure™7.5, consisting of approximately 75 wt % Rebaudioside A and 25 wt %stevioside.

Preference Testing

Paired comparison testing was conducted for sweetness and preference ofblends of BlendSure 7.5 and Sample A having sweetness equal to 97%Rebaudioside A in a pH 3 citric acid buffer (0.045% citric acid and0.013% sodium citrate) with a panel of taste testers. The tests wereconducted as complete block designs with between 24 to 46 evaluations.The presentation order was rotated. The solutions were served in 2 ouncesoufflé cups labeled with 3-digit codes at room temperature. Thepanelists were instructed to consume at least half of each sample. Therewas a one minute enforced waiting period between tests to clear thepanelists' palates. The panelists were asked to identify the solutionthat was sweeter and which they liked better. Bottled water, 2% sucrosesolution, and unsalted crackers were available for the panelists toclear their palates before and during testing. The sweetness resultswere analyzed as two-tailed tests at an alpha risk of 0.05 with thebinomial test, as shown below.

The results of the sweetness and preference questions were analyzed withthe binomial test and the Thurstonian d′ calculated. The p-value for aone-tailed binomial test is calculated as

$1 - {\sum\limits_{k = 0}^{c}\; {\begin{pmatrix}n \\k\end{pmatrix}{p_{0}^{k}( {1 - p_{0}} )}^{n - k}}}$

where c is the number of successes, n is the number of trials, and p₀ isthe chance probability. A test is considered statistically significantwhen the p-value is less than the a priori set alpha risk. Thetwo-tailed p-value is double the one-tailed p-value as calculated above.

Thurstonian d′ is a linear measure of psychophysical difference. A d′=1is generally considered to be a just-noticeable-difference (JND) where astimulus will be judged stronger in 75% of the trials. The Thurstoniand′ is independent of test method and for paired comparison tests iscalculated as

p _(c)=Φ(d′/√{square root over (2)})

where p_(c) is the proportion of successes, and Φ(·) is the cumulativedistribution function of the standard normal distribution. A completetreatment of these statistical calculations can be found in standardtextbooks on the subject (Bi J., “Sensory Discrimination Tests andMeasurements,” Blackwell Publishing, 2006, Chapters 2 and 9).

Combined replicated test results are shown below in Table 1.

TABLE 1 Samples Preference Sweetness Ppm Blend Ppm Blend Fraction Ppmcontrol Reb A Blend p-value Reb A Blend p-value Sample A BlendSureSample A Reb A Count count one-tailed count count two-tailed 444 2960.60 605 22 18 0.68 16 24 0.15 547 365 0.60 705 13 27 0.01 15 25 0.08547 365 0.60 800 22 46 <0.01 24 44 0.01 547 365 0.60 900 28 40 0.06 4523 0.00 660 440 0.60 900 17 27 0.05 15 29 0.02 660 440 0.60 1000 18 260.09 23 21 0.65 675 225 0.75 900 9 31 <0.01 24 16 0.15 750 250 0.75 100012 28 <0.01 26 14 0.04 825 275 0.75 1000 8 38 <0.01 24 22 0.66

In pH 3 citric acid buffer and at sweetness levels that were notsignificantly different (p-value >0.05 two tailed), the above dataindicate a preference for blends containing ratios of Mogroside V toRebaudioside A within certain ranges. Specifically, the following can beseen.

A blend of 675 ppm Sample A and 225 BlendSure 7.5 (75% Sample A, 900 ppmtotal) was significantly preferred over 900 ppm 97% Rebaudioside A.

A blend of 825 ppm Sample A and 275 BlendSure 7.5 (75% Sample A, 1100ppm total) was significantly preferred over 1000 ppm 97% Rebaudioside A.

A blend of 444 ppm Sample A and 296 BlendSure 7.5 (60% Sample A, 740 ppmtotal) was not significantly different from 605 ppm 97% Rebaudioside A.

A blend of 547 ppm Sample A and 365 BlendSure 7.5 (60% Sample A, 912 ppmtotal) was significantly preferred over 705 ppm 97% Rebaudioside A.

A blend of 660 ppm Sample A and 440 BlendSure 7.5 (60% Sample A, 1100ppm total) was not significantly different in preference from 1000 ppm97% Rebaudioside A.

Blends of Sample A and BlendSure 7.5 that contained 75% Sample Aperformed better than blends containing 60% Sample A against 97%Rebaudioside A.

Blends of Sample A and BlendSure 7.5 were more preferred over 97%Rebaudioside A as the level of sweetness increased.

Example 2 Stevia-Mogroside V Blends Vs. Stevia Preference Testing

Blends of a solid Luo Han Guo extract containing 50 wt % Mogroside Vwith a Stevia product containing mostly Rebaudioside A in a pH 3 citricacid buffer (0.045% citric acid and 0.013% sodium citrate) were comparedin sweetness and preference panel testing against the Stevia product.The blends and the Stevia product were as described in Example 1.Synergy can be detected by the construction of isoboles (iso-effectcurves) where the concentrations of two substances that have equaleffect, in this case sweetness, are plotted on a chart with the axisbeing the concentration of the substances. Linear isoboles result whenthere is no synergy between the two substances. An isobole with adownward curvature results when there is synergy between the twosubstances. A complete discussion of isoboles and synergy can be foundin Berenbaum, “What is Synergy”, Pharmacological Reviews, Vol. 1989, No.41 pages 93-129. Blends of BlendSure 7.5 and Sample A having sweetnessequal to 500 ppm, 700 ppm, and 900 ppm BlendSure 7.5 were predicted fromlinear isoboles with the assumption of no sweetness synergy.

Paired comparison testing was conducted for sweetness and preference ofblends of BlendSure 7.5 and Sample A that are equal sweet to levels of500 ppm, 700 ppm, and 900 ppm BlendSure 7.5 with a panel of tastetesters. The tests were conducted as complete block designs with between34 to 44 evaluations. The presentation order was rotated. The solutionswere served in 2 ounce soufflé cups labeled with 3-digit codes at roomtemperature. The panelists were instructed to consume at least half ofeach sample. There was a one minute enforced waiting period betweentests to clear the panelists' palates. The panelists were asked toidentify the solution that was sweeter and which they liked better.Bottled water, 2% sucrose solution, and unsalted crackers were availablefor the panelists to clear their palates before and during testing. Theresults were analyzed as in Example 1, and are summarized in Table 2.

TABLE 2 Samples Preference Sweetness Ppm Blend Ppm Blend Fraction Ppmcontrol BlendSure Blend p-value BlendSure Blend p-value Sample ABlendSure Sample A BlendSure count count one-tailed count counttwo-tailed 133 399 0.25 500 38 43 0.51 44 37 0.37 284 284 0.50 500 32 490.04 41 40 0.82 458 153 0.75 500 13 31 <0.01 20 24 0.45 660 0 1.00 50029 52 0.01 35 46 0.18 179 537 0.25 700 33 46 0.11 50 29 0.01 367 3670.50 700 23 56 <0.01 33 46 0.11 563 188 0.75 700 23 56 <0.01 38 41 0.65770 0 1.00 700 32 47 0.07 43 36 0.37 226 679 0.25 900 24 48 <0.01 39 330.41 455 455 0.50 900 22 50 <0.01 34 38 0.56 686 229 0.75 900 23 49<0.01 40 32 0.29 920 0 1.00 900 19 53 <0.01 37 35 0.72

As can be seen from the above data, there was no evidence of sweetnesssynergy between BlendSure 7.5 and Sample A. However, the preference fora blend of BlendSure 7.5 and Sample A over BlendSure 7.5 alone increasedas the ratio of Sample A to BlendSure 7.5 increased, and also assweetness increased.

Example 3 Removal of Aromatic Glycosides and Semi-Volatile OrganicCompounds from Luo Han Guo Extract

A 3′× ½″ ID jacketed glass column (Ace glass incorporated) ofapproximately 115 ml of capacity was packed with approximately 57 g ofvirgin granular active carbon (CAL 12×40 from Calgon Corporation) thathad been freshly washed with boiling water. The column jacket was heatedto 60° C. and held at that temperature during the duration of theexperiment. After packing the column, approximately 500 mL of deionizedwater was passed through the carbon bed at a flow rate of 2.5 mL/min inorder to displace and remove carbon lines. A 27% % wt solution ofBiovittoria Fruit Sweetness™ (approximately 50 wt % Mogroside V drysolids basis (dsb)) was prepared by dissolving 1.241 kg of BiovittoriaFruit Sweetness™ in 3.318 kg of Milli-Q water (water provided by aMilli-Q reverse osmosis water purification system, available fromMillipore Corp.). The solution was then heated to 60° C., passed througha Millipore Optiseal Durapore 0.22 μm hydrophilic pleated cartridgefilter to a sterile feed bottle and held at 60° C. during the run.

The solution was passed through the column at a rate of 2.6 g/min(equivalent to 1.25 Bed volumes per hour) using MASTERFLEX® tubing 13and a peristaltic pump (MASTERFLEX® pump). The effluent was collected in90 minute fractions, with an average mass of 234 grams per fraction.After fraction 19 (elapse run time: 28.5 hours), the effluent was tastedand it was informally determined that the effluent had a significantlybetter taste than the feed material. Therefore, an additional 355.5 g ofBiovittoria Fruit Sweetness™ was dissolved in 945.3 g of Milli-Q water,brought up to 60° C., passed through a Millipore Optiseal Durapore 0.22μm hydrophilic pleated cartridge filter, and added to the feed bottle.After 37.5 hours, the sweeten-off (the term “sweeten off” is understoodas washing the column for displacement of remaining Fruit Sweetness™solution) was started by changing the column feed to Milli-Q water (@60° C.). The column was allowed to sweeten off for 6 hours until therefractive index of the effluent was similar to that of water. Thematerial collected in all fractions corresponded to an overall massyield of approximately 98 wt % of the dry material fed to the apparatus.The treatment level of the total material feed to the apparatus (1241g+335 g) was calculated to be 3.61 wt %. A Roundtable of eightexperience tasters compared acceptability in regard to reducedoff-flavor by comparing water solutions of Fruit Sweetness™, Compositeof fractions 1 through 5, Composite of fractions 1 through 10, Compositeof fractions 1 through 15, Composite of fractions 1 through 20, andComposite of fractions 1 through 25. It was found that the Composites of1 through 5, 1 through 10 and 1 through 15 presented a significant levelof taste improvement over the Fruit Sweetness™ feed material. AComposite of fractions 1 through 15 contained a dry mass of 947 g, thuscorresponding to a carbon treatment level of 6.0 wt %. The Composite offractions 1 through 15 was henceforth identified as SAMPLE A (286683).

HPLC was used to determine the aromatic glycoside composition of theBiovittoria Fruit Sweetness™ feed and better tasting Luo Han Guoeffluent after carbon treatment. A Waters 2695 Separations Module wasequipped with a Waters 2487 Dual λ Absorbance Detector and a PhenomenexGemini C18 Column, 5 μm, 150×4.6 mm with Phenomenex Gemini C18 SecurityGuard cartridge, 4×3 mm. An acetonitrile/water gradient listed below wasused as the mobile phase, at a flow rate of 10.0 mL/min and a columntemperature of 40° C. UV detection at 203 nm was used, and the injectionvolume of 40 μL.

Mobile Phase: Acetonitrile/Water volume % linear segment gradient Time[min] Acetonitrile H₂O 0 20 80 15 30 70 20 50 50 25 50 50 30 20 80

A pure Mogroside V standard (ChomaDex, Inc.) was used for calibratedquantitation of all components detected at 203 nm. Table 3 summarizesthe wt % of components on a dry solids basis (d.s.b) as Mogroside V. Asignificant reduction of aromatic glycosides eluting from the HPLCcolumn in the range of 3.5 to 4.5 minutes under the above definedconditions was observed between the Biovittoria Fruit Sweetness™ feedand the carbon effluent, and this reduction corresponded to the as notedsignificant flavor improvement.

TABLE 3 Aromatic Aromatic Mog V wt % of glycosides wt % glycosides wt %total sample of total sample relative to Sample ID dsb as Mog V Mog VBiovittoria Fruit 49.1% 7.3% 14.8% Sweetness ™ feed (284178) Luo Han Guo50.9% 4.2% 8.3% Sample A (286683)

Headspace GC with Flame Ionization detection (FID) as defined by thefollowing conditions was used to determine the composition ofsemi-volatile organic compounds in the Luo Han Guo feed and the bettertasting Luo Han Guo recovered after carbon treatment.

Combi PAL Autosampler

Mode: Headspace

Syringe Volume: 1 mL

Syringe Temperature: 85° C.

Agitator Temperature: 80° C.

Pre-incubation Time: 30 minutes

Pre-incubation Agitator Speed: 500 rpm (5 sec on, 2 sec off)

Plunger Fill Speed: 200 μL/sec

Viscosity Delay: 12 sec

Pre-injection Delay: 0 sec

Plunger Inject Speed: 100 μL/sec

Post-injection Delay: 10 sec

Syringe Flush Time: 3 min

GC Cycle Time: 54 min

Varian 3800 GC

Oven:

Initial Temperature: 40° C.

Initial Hold Time: 5 minutes

Ramp: 7.5° C./min

Final Temperature: 235° C.

Final Hold Time: 14 minutes

Front Inlet (1177):

Temperature: 250° C.

Mode: Splitless

Column:

Type: Rtx-624 (30 m×0.25 mm×1.4 μm) Restek Cat #10968

Mode: Constant Flow

Flow: 1.0 mL/min (Helium)

Middle Valve Oven:

Temperature: 250° C.

Varian 4000 FID

Temperature: 250° C.

Makeup gas: 2 mL/min (He)

H₂ flow: 40 mL/min

Air flow: 450 mL/min

Varian 4000 Ion Trap MS

Scan Type: Full

Mass Range: 25-275 m/z

Scan Time: 0.00 to 45.00 minutes

Ionization Type: EI

Target TIC: 20000 counts

Max Ion Time: 25000 μsec

Emission Current: 10 μamps

Scans Averaged: 3 μscans (0.60 sec/scan)

Data Rate: 1.67 Hz

Multiplier Offset: 0 V

A pure D-limonene standard (Sigma-Aldrich) was used for calibratedquantitation of all semi-volatile organic compound components shown inFIG. 3. Table 4 summarizes the total semi-volatile organic compounds ppmwt of components on a dry solids basis (dsb) as D-limonene. Asignificant reduction of semi-volatile organic compounds fromBiovittoria Fruit Sweetness™ feed (284178) to carbon treated Luo Han Guo(Sample A 286683) is seen, corresponding to the significant improvementof Luo Han Guo flavor.

TABLE 4 Semi-volatile Semi-volatile organic organic compounds compoundsMog V wt % ppm wt as ppm wt of total D-limonene of relative to Sample IDsample dsb total sample Mog V Biovittoria Fruit 49.1% 8.9 18 Sweetness ™feed (284178) Luo Han Guo 50.9% 0.6 1.2 Sample A (286683)

Example 4 Identification of Off-Flavor Components in Luo Han Guo Extract

Sensory evaluation has found that Luo Han Guo material that has passedthrough carbon in aqueous solution has a better, more acceptable flavorthan the feed Luo Han Guo material. HPLC analysis of an exemplary dryLuo Han Guo extract having a Mogroside V content of about 50 wt %(Biovittoria Fruit Sweetness™), and analysis of the same material thathad been carbon treated and spray dried, are shown in the upper andlower chromatograms respectively in FIG. 1. The HPLC method parameterswere as in Example 3 with the following modified linear segmentgradient.

Mobile Phase: Acetonitrile/Water volume % linear gradient Time [min]Acetonitrile H₂O 0 10 90 10 10 90 20 20 80 25 20 80 30 30 70 35 30 70 5595 5 65 95 5 75 10 90

HPLC analysis showed that the profile of mogroside isomers remainedessentially unchanged after carbon treatment. An enlarged view of themore polar region of the chromatograms of FIG. 1 is shown in FIG. 2,where the treated product shown in the lower chromatogram shows peaks inthe vicinity of 21 min (marked with an arrow) greatly decreased relativeto the untreated product in the upper chromatogram. In order todetermine the relationship of the component(s) eluting near 21 min andthe decrease in “musty” off-flavor, a series of extraction andpurification steps was applied to spent carbon that had been used totreat Luo Han Guo in a manner similar to Example 3. After eachextraction purification step throughout this study, an expert panel oftasters evaluated the samples of carbon treated Luo Han Guo that hadbeen spiked with ˜5-10× of the original level of the recoveredcomponents in order to identify the samples which exhibited thecharacteristic “musty” off-flavor of Fruit Sweetness™.

To recover components removed by carbon treatment of aqueous Luo Han Guosolution approximately 500 g of spent carbon that had been used for LuoHan Guo refinement was sequentially extracted with multiple 350 mLaliquots of solvents after water washing. Ethanol and then acetone wereused to wash the carbon. The extracts were filtered through a 0.45 μmnylon filters and evaporated to dryness under a stream of nitrogen atambient temperature to recover approximately 2.0 g of solid. The residuefrom the acetone extract was observed by the aforementioned expert panelof tasters to contain the significant “musty” off-flavor characteristicof Fruit Sweetness™. It was also confirmed that the 21 min HPLCcomponent (FIGS. 1 and 2) was contained in the acetone fraction and alsoin all subsequent “musty” off-flavor fractions, as described below.

Liquid-liquid extraction between 50 mL water and 50 mL chloroform wasapplied to the initial acetone extracted fraction after complete drying.The “musty” off-flavor remained with the water soluble fraction(approximately 1.8 g of solid recovered). Subsequently, solid phaseextraction (SPE) using four stacked Waters Sep-Pak C18 SPE cartridges(Waters Corp., WAT020515) was applied to further fractionate theoff-flavor residue. Approximately 10 mg/mL residue in water was loadedonto the cartridges 10 mL at a time after conditioning the SPEs with 5mL of methanol and 10 mL of Milli-Q water. Recovered fractions were thenobtained using a series of 10 mL SPE washes as follows; 100% water, 2%acetonitrile (MeCN)/98% water, 5% MeCN, 10% MeCN, 20% MeCN, 25% MeCN,30% MeCN, 40% MeCN, 50% MeCN, and 100% MeCN. All extracts were driedunder a stream of nitrogen and evaluated by a sensory panel. This entireisolation procedure from spent carbon through to SPE fractionation wasrepeated three times with the same sensory results.

The characteristic “musty” off-flavor was determined to be significantlyconcentrated in the approximately 250 mg of solids recovered from the20% MeCN/80% water eluted SPE fraction (288054) as verified by theexpert panel of tasters when spiked into a water solution of carbontreated Luo Han Guo. HPLC of this fraction again showed the 21 mineluting component, FIG. 4, Chemical analyses of the primary componentsof this isolate, relative to an SPE blank (10 mL water+10 mL 50% MeCN+10mL 100% MeCN) were conducted using; Antek total nitrogen,Folin-Ciocalteu phenolic colorimetric test, ninhydrin proteincolorimetric test, ion chromatography amino acid analysis of an aciddigest, ATR-FTIR of dry solid, LC-MS, and NMR. Results are summarized inTables 5 and 6, and are consistent with an aromatic glycoside class ofcompounds,

TABLE 5 Test 288054 - SPE 20% MeCN Folin-Ciocalteu phenolics expressed17.6 mg/mL as gallic acid equivalents Ninhydrin protein color testYellow (minimal protein) Antek total nitrogen 0.4% wt IC Amino acids2.4% wt as protein ATR-FTIR FIG. 5 LC-MS FIG. 6 H-NMR, ¹³C-NMR, COSY-45,Table 5 DEPT-135

FIG. 5 shows the baseline corrected ATR-FTIR. spectrum of fraction288054. Characteristic bands for OH, aliphatic CH, CO, and weak phenylabsorbances can be seen, all consistent with the presence of an aromaticglycoside. No C═O absorbance is observed.

FIG. 6 shows the Time-of-Flight (ToF) accurate Mass Spectrum for themajor component corresponding to sample 288054 in FIG. 4 with retentiontime of 21.0 minutes. The inserted table in FIG. 6 lists the mostprobable stoichiometric formula for mass ion 503 Daltons. The mostprobable accurate mass with 1.6 ppm mass accuracy is shown to be aC₂₆H₃₀O₁₀ neutral charge compound.

TABLE 6 NMR measurement for Structural sub-unit sample 288054 NMR shiftresonances information ¹H-NMR 5.6 ppm and 5.1 ppm doublets Glycosidesub-units typical on anomeric protons; multiple resonances between 3.9ppm and 3.3 ppm ¹H-NMR multiple resonances between Substituted aromatic6.85 to 7.10 ppm rings COSY-45 Connectivities consistent with glycosidicproton resonances COSY-45 3.75, 3.8 ppm Methoxy subunits ¹³C/DEPT-135resonances for methines 101, Glycoside sub-units 75 to 70 ppm; andmethylenes 61 to 63 ppm. ¹³C/DEPT-135 resonances for methines 119,Substituted aromatic 116, 111 ppm; rings; phenoxy methyl resonanceAromatic methoxy 53.7 ppm sub-units

The distribution and suggested identity of a variety of semi-volatilecomponents was evaluated using headspace of 5% aqueous solutions via GasChromatography with mass spectrometric detection (GC-MS) as defined inExample 3. FIG. 3 shows a comparison of the semi-volatile componentprofile of Fruit Sweetness™ feed (284178) and Sample A. Table 7 shows alisting of best MS library matches for 28 semi-volatile organiccompounds corresponding to those labeled in FIG. 3. Known flavor andodor organoleptic responses to these compounds are listed for comparison(see for example, Mosciano, G., Perfumer and Flavorist 25, No. 6, 26,(2000).

TABLE 7 Peak ref. (FIG. 3) Component name (CAS#) Known organolepticresponse A 2-pentyl-furan (3777-69-3) Green, waxy, with musty, cookedcaramellic nuances B Butyl butanoate (109-21-7) Sweet, fresh, fruity,slightly fatty C D-limonene (5989-27-5) Sweet, orange, citrus and terpyD t-butylbenzene (98-06-6) — E Gamma-terpinene (99-85-4) Terpy, citrus,lime-like, oily, green with a tropical fruity nuance F Butyl butenoate(7299-91-4) — G Terpinolene (586-62-9) Citrus, Lime, Pine, plasticNonanal (75718-12-6) — H Durene (95-93-2) — I 1,3,8-p-menthatriene(21195-59-5) — J p-cymene (99-87-6) Terpy and rancid with slightly woodyoxidized citrus notes K Hexyl butyrate (2639-63-6) Apple, Fruity, Green,Soapy, Sweet L 1,1,5,6-Tetramethylindane — (942-43-8) M Azulene(275-51-4) — N α-ionene (475-03-6) — O 1-methyl-naphthalene (90-12-0)Naphthyl-like with a medicinal nuance P 2-methyl-naphthalene (91-57-6) —Q dehydro-ar-ionene (30364-38-6) licorice R (−)-α-Cedrene (469-61-4)woody cedar S Z-β-farnesene (28973-97-9) citrus green T (+)-β-Cedrene(546-28-1) — U Trans-α-bergamotene (13474-59-4) woody V1cis-α-bisabolene (29837-07-8) — V2 α-farnesene (502-61-4) Fresh greenvegetative, with celery and hay nuances and somewhat fatty and tropicalfruity afternotes W (−)-β-bisabolene (495-61-4) balsamic X(+)-α-Longipinene (5989-08-2) — Y 2-hexyl-1-dodecanol (2425-77-6) — Z(E)-Nerolidol (40716-66-3) green floral woody fruity citrus melon

Example 5

An amount of 40 g of Fruit Sweetness™ was dissolved in 200 g of Milli-Qwater in a 500-mL beaker and 30 g of activated carbon (BG-HHM fromCalgon Carbon Corporation) was added to the Fruit Sweetness™ solution.The activated carbon slurry was stirred for 2 hours, while taking 50 μLsamples of sterile filtered solution at 0, 5, 15, 30, 60, 90, and 120minutes. The samples were diluted 20-fold in Milli-Q water and analyzedby HPLC for relative abundance of mogrosides between time points. After2 hours, the activated carbon slurry was filtered through Whatman #2filter paper and the filtrate was sterile filtered into a tared freezedrying bottle. Once the sterile filtrate had been freeze dried, its masswas recorded and analyzed with HPLC for Mogroside V content. Thefreeze-dried material was designated Sample B. A 550 ppm neutral pHwater solution of Sample B (carbon slurry treated Fruit Sweetness™) wasthen tested against 500 ppm Reb A 97 in neutral pH water for tastepreference by 48 to 50 panelists. For comparison, the Fruit Sweetness™was also tested against 500 ppm Reb A 97 for preference in neutral pHwater. The tests were conducted as complete block designs. Thepresentation order was rotated. The solutions were served in 2 ouncesoufflé cups at room temperature. The panelists were instructed toconsume all of the sample. The panelists were not allowed to retaste thesamples. The panelists were asked to identify the solution that wassweeter and which they preferred. Bottled water, 2% sucrose solution,and unsalted crackers were available for the panelists to clear theirpalates before and during testing.

The data were analyzed with the binomial test with an alpha risk of 0.05as two-tailed tests for sweetness and a one-tailed test for preference.

TABLE 8 Preference Sweetness One-tailed Two-tailed count p-value countp-value 500 ppm Reb A 97 22 0.24 22 0.47 550 ppm Fruit 26 26 Sweetness ™

TABLE 9 Preference Sweetness One-tailed Two-tailed count p-value countp-value 500 ppm 6 <0.01 21 0.20 Reb A 97 550 ppm 44 29 Sample B

Table 8 shows that the commercial product Fruit Sweetness™ was notsignificantly preferred over Reb A at equi-sweetness level. However, theslurry carbon-treated Fruit Sweetness™ Sample B was significantlypreferred over BlendSure 7.5 at equi-sweetness level (Table 9).

Although the invention is illustrated and described herein withreference to specific embodiments, the invention is not intended to belimited to the details shown. Rather, various modifications may be madein the details within the scope and range of equivalents of the claimswithout departing from the invention.

What is claimed is:
 1. A method of purifying a Luo Han Guo extractcomprising contacting the Luo Han Guo extract with activated carbon,wherein the Luo Han Guo extract that is contacted is one that haspreviously been contacted with at least one of an ion exchange resin ora macroporous polymeric adsorbent resin.
 2. The method of claim 1,wherein the Luo Han Guo extract that is contacted is one that haspreviously been contacted with a macroporous polymeric adsorbent resin.3. The method of claim 1, wherein the Luo Han Guo extract that iscontacted is one that has been previously contacted with both amacroporous polymeric adsorbent resin and an ion exchange resin.
 4. Themethod of claim 1, wherein the Luo Han Guo extract that is contacted isone that has been previously contacted with first a macroporouspolymeric adsorbent resin and then an ion exchange resin.
 5. The methodof claim 1, wherein the Luo Han Guo extract is in the form of an aqueoussolution and the aqueous solution is passed through a column of granularactivated carbon.
 6. The method of claim 1, wherein the activated carbonis in the form of powder, granules or beads.
 7. The method of claim 1,wherein the Luo Han Guo extract that is contacted is one that haspreviously been contacted with an ion exchange resin that is an anionicresin.
 8. The method of claim 1, wherein the Luo Han Guo extract iscontacted with an amount of activated carbon for a contact timeeffective to reduce the levels of aromatic glycoside and semi-volatileorganic compound impurities present in the Luo Han Guo extract.
 9. Themethod of claim 1, wherein the Luo Han Guo extract contacted withactivated carbon is in the form of an aqueous solution.
 10. The methodof claim 1, wherein the Luo Han Guo extract contacted with activatedcarbon has been prepared by dissolving a powdered dry extract of Luo HanGuo fruit in water to obtain a dissolved extract.
 11. The method ofclaim 10, wherein the dissolved extract is subjected to microfiltration.12. The method of claim 1, wherein the activated carbon has a surfacearea of over 100 m²/g.
 13. The method of claim 1, wherein from 2 wt % to15 wt % active carbon relative to Luo Han Guo extract on a solids basisis used.
 14. The method of claim 1, wherein the Luo Han Guo extract isin the form of an aqueous solution, the aqueous solution is passedthrough a column of granular activated carbon to obtain an effluent, andthe effluent is subsequently concentrated.
 15. The method of claim 14,wherein the effluent is concentrated by evaporation or membraneconcentration.
 16. The method of claim 14, wherein the effluent isconcentrated by membrane concentration using a nanofiltration membraneor a reverse osmosis membrane.
 17. The method of claim 14, whereinfollowing concentration the effluent is dried.
 18. The method of claim17, wherein the effluent is dried using a spray drying unit or a sprayagglomeration unit.
 19. The method of claim 14, wherein followingconcentration the effluent is combined with one or more componentsselected from the group consisting of Rebaudioside A, Rebaudioside B,Rebaudioside D, Steviol glycoside and purified Stevia extract and thendried.
 20. The method of claim 1, wherein contacting the Luo Han Guoextract with activated carbon removes pesticide residues from the LuoHan Guo extract.
 21. The method of claim 1, wherein water is the onlycarrier present during the contacting of the Luo Han Guo extract withthe activated carbon.
 22. A method of purifying a Luo Han Guo extractcomprising a) contacting the Luo Han Guo extract sequentially with amacroporous polymeric adsorbent resin, an anionic ion exchange resin andactivated carbon, wherein an aqueous solution of the Luo Han Guo extractis passed through a column of granular activated carbon having a surfacearea of over 100 m²/g and is contacted with an amount of granularactivated carbon for a contact time effective to reduce the levels ofaromatic glycoside and semi-volatile organic compound impurities andpesticide residues present in the Luo Han Guo extract, thereby obtainingan effluent, and b) drying the effluent using a spray drying unit or aspray agglomeration unit.